(A) Selection of quiescent bone marrow cells using 5-FU. Bone marrow mononuclear cells were incubated for 7 days in IMDM either supplemented with IL-3, SCF, and 5-FU to kill dividing cells (right-hand panels) or not (left-hand panels). At 4 days of incubation an aliquot was spun onto microscope slides and stained with Wright’s stain (top panels). Twenty-four hours before harvesting, 3H thymidine was added to the cultures. Following 7 days incubation cells were obtained and spun onto microscope slides. The slides were dipped in photographic emulsion and exposed for 7days before being developed, counterstained with Wright’s stain, and visualized under the light microscope (middle panels). (Bottom panels) Cells as above, but stained with antibody to c-kit and viewed under fluorescence. (B) Long-term culture. 5-FU–selected cells were plated on monolayers of bone marrow derived fibroblast as described in Materials and Methods and used to initiate long-term bone marrow cultures. The arrows show areas of extensive lipid deposition characteristic of these types of culture. Arrowheads identify “cobblestone areas,” clusters of developing hematopoietic cells.
